The overall goal of these studies is to determine the mechanism(s) underlying opioid-mediated signaling in immune suppression. Physical and psychological stress has profound effects on the immune system of humans and animals. Stress is also a known risk factor for many human diseases, such as infectious and autoimmune diseases. Studies from our laboratory and others support the hypothesis that stressors modulate immune function through stress hormones such as endogenous opioids, other than exclusively glucocorticoids. We have reported that physical stress induces lymphocyte apoptosis in an endogenous opioid-dependent manner. However, the mechanisms by which stress affects the immune system remain to be elucidated. During the last grant period, we reported that phosphatidylinositol 3-kinase (PI3K)/Akt signaling is involved in immune responses following stress. Our in vitro studies have shown that opioids prime lymphocyte apoptosis via PI3K. Our preliminary results showed that chronic stress inhibits T cell mediated immune responses. Moreover, we found that 5 opioid receptor (MOR) is a novel receptor in stress-induced lymphocyte apoptosis. Our in vitro studies showed that glycogen synthase kinase 32 (GSK32) is required for MOR-mediated apoptosis. We and others revealed that the function of MOR is regulated by 2-arrestin 2, a multifunctional adaptor and signal transducer. Our in vitro results showed that 2-arrestin 2 inhibits lymphocyte apoptosis through p38 mitogen-activated protein kinase (MAPK). In addition, we also found that 2-arrestin 2 plays a role in stress-induced lymphocyte reduction. However, the mechanisms by which MOR and 2-arrestin 2 contribute to stress-induced immune suppression are unclear. Our hypothesis is that MOR and 2-arrestin 2 mediated pathways are critical to immune suppression. Moreover, we postulate that the mechanisms involve the MOR-mediated PI3K and GSK32 pathways, and 2-arrestin 2 mediated p38 MAPK signaling. To test this hypothesis, we will pursue two aims. Aim 1 will examine the role of PI3K and GSK32 in MOR- mediated immune suppression. We will treat MOR knockout mice and wild type mice with or without PI3K/GSK3 inhibitors, and then subject them to physical stress for different time periods. We will first define the role of MOR-mediated PI3K signaling pathway in immune suppression induced by stress, and next examine the effects of MOR-mediated GSK32 signaling on stress-induced lymphocyte apoptosis. Aim 2 will define the contribution of p38 MAPK in 2-arrestin 2-mediated immune suppression. 2-arrestin 2 knockout mice and wild type mice will be administrated p38 MAPK inhibitor, and then subjected to physical stress to investigate role of 2-arrestin 2 mediated p38 MAPK signaling in immune suppression following stress. In addition, the effects of 2-arrestin 2 on a developing and an established immune response will be examined. These studies should lead to our understanding of the effects of opioid-mediated signaling on the immune system, providing the opportunity to develop novel strategies and therapeutics for immune suppression. PUBLIC HEALTH RELEVANCE: Psychological and Physical stress can have pronounced effects on the immune system. Historically this affect was attributed to the release of stress hormones although other factors appear to play an important role. Using a mouse model of physical restraint stress we have investigated some of the molecular mediators which may account for the connection between stress and the immune system. This research will examine the mechanisms whereby the immune system is influenced by stress with the hope of leading to the development of specific treatments or drugs which in turn may ameliorate this sometimes deleterious interaction.